The receptor for erythropoietin, EpoR, is a homodimeric cytokine receptor essential for the formation of red cells. In addition, it is the principal homeostatic regulator of red cell production rate, through a wide dynamic range. The molecular mechanisms that encode quantitative features of EpoR signaling are largely unknown, but may involve the control of apoptosis in early erythroid progenitors. Dysregulation of these mechanisms may lead to oncogenic transformation. The purpose of this proposal is to identify molecular mechanisms that encode the quantitative response to EpoR signaling, and whose dysregulation predisposes to leukemia. The following approaches will be taken: 1. The receptor for prolactin, PrlR, a non-hematopoietic cytokine receptor, can rescue EpoR-/- progenitors and support their full differentiation into red cells in vitro. This and similar findings suggest that homodimeric cytokine receptors signal through a similar, 'generic' program. Components of this program will be identified using in-vitro red-cell differentiation assays. These may be relevant to other cytokine- receptor regulated systems such as mammary epithelium 2. In spite of apparent functional similarity, PrlR and EpoR share little homology in their cytoplasmic signaling domains. Gene targeting in mice will be used to generate 'knock-in' models that allow stringent testing in vivo of as yet unsuspected adaptations by these. The hypothesis that these two receptors differ in their quantitative regulation of tissue homeostasis will be tested. 3. A novel flow-cytometry assay that allows analysis and separation of erythroid precursor populations at specific maturation stages from freshly explanted hematopoietic tissue is currently being developed by the applicant. Together with expression microarrays and other approaches, this will be used to identify EpoR gene targets and cellular functions that modulate erythropoietic rate. This approach was recently validated by looking at mice mutant for the EpoR-activated transcription factor Stat 5. This identify the anti-apoptotic Stat5-bcl-xL pathway in early erythroblasts as a determinant of erythropoietic rate. 4. Retroviral expression cloning will be used to identify new erythroid anti-apoptotic genes. Components of the apoptotic machinery will be assessed as candidate modulators of erythropoietic rate and/or leukemogenic transformation.